1. Field of the Invention
This invention relates to synthetic methods for synthesizing site-specifically radiolabeled oligonucleotides.
2. Summary of the Related Art
Recently, considerable interest has been generated in the development of synthetic oligonucleotides as therapeutic or gene expression modulating agents in the so-called antisense approach. For example, Agrawal, Trends in Biotechnology 10, 152-158 (1991), extensively reviews the development of antisense therapeutic approaches. Oligonucleotide phosphorothioates (PS-oligos) have shown great therapeutic potential as antisense-mediated inhibitors of gene expression (Stein and Cheng, Science 261, 1004 (1993), and references therein) as evidenced by a number of ongoing clinical trials against AIDS and cancer. Agrawal and Tang, Antisense Res. and Dev. 2, 261 (1992), and references therein; and Bayever et at., Antisense Res. Dev. 3, 383 (1993).
For an antisense therapeutic approach to be effective, oligonucleotides must be introduced into a patient and must reach the specific tissues to be treated. The biodistribution and pharmacokinetics of a therapeutic drug must be determined as a step preliminary to treatment with the drug. Consequently, there is a need to be able to detect oligonucleotides in body fluids or tissues. Agrawal et al., Clin. Pharmacokinetics 28, 7 (1995), reviews certain aspects of the pharmacokinetics of antisense oligonucleotides.
Detection of specific nucleic acid sequences present in cells is generally known in the art. Southern, J. Mol. Biol. 98, 503-517 (1975) teaches detection of specific sequences among DNA fragments separated by gel electrophoresis using "blotting" or transfer of the DNA fragments to a membrane followed by hybridization of denatured DNA fragments with a radioactive probe and autoradiography. This procedure has also been extended to the detection of RNA molecules extracted from cells or tissues. E.g., Sambrook et al., Molecular Cloning: A Laboratory Manual pp. 7.37-7.52 (Cold Spring Harbor Laboratory Press, 2d Ed. 1989). More recently, faster and quantitative "dot-blotting" procedures have been developed for rapid detection of DNA or RNA from tissues or cells. PCT Application WO 94/16103 discloses a method for detecting the presence of unlabeled synthetic oligonucleotides in body fluids or tissue samples taken from laboratory animal and human patients. In that method, body fluid or tissue samples are taken from an animal or human to whom an oligonucleotide has been administered and are proteolytically digested, then extracted. Total nucleic acids are then transferred to a hybridization membrane. The hybridization membrane with attached nucleic acids is prehybridized, then hybridized with a labeled oligonucleotide that is complementary to the oligonucleotide that was administered to the animal or patient. Presence of hybridized, labeled oligonucleotide is then detected by standard procedures.
Another well-established approach used in in vivo pharrnacokinetic studies of pharmacological compounds such as antisense oligonucleotides entails radiolabeling the compounds to enable detection. In animal models, radiolabeled oligonucleotides have been administered to the animal and their distribution within body fluids and tissues has been assessed by extraction of the oligonucleotides followed by autoradiography (See Agrawal et at., Proc. Natl. Acad. Sci. 88, 7595-7599 (1991).
.sup.35 S is a common iostopic label used to study the pharmacokinetics and biodistribution of drug compounds. .sup.35 S-labeling is an established and wide-spread technique. For biological studies, .sup.35 S-labeled oligonucleotide phosphorothioates have been prepared using H-phosphonate chemistry. Garegg et al., Chem. Scr. 25, 280-282 (1985). Recently, Iyer et at., Tel. Lett. 35, 9521-9524 (1994), disclosed a new compound and methods for synthesizing .sup.35 S site-specifically-labeled oligonucleotide phosphorothioates.
.sup.14 CC and .sup.3 H are two other commonly used isotopic labels. Radioisotopic labeling of synthetic oligonucleotides with .sup.14 C and .sup.3 H is currently accomplished by using the well-established solid-phase automated synthesis. Beaucage and Caruthers, Tetrahedron Lett. 22, 1859-1862 (1981); International Application PCT/US85/01148; and Barone et at., Nucleic Acids Res. 12, 4051 (1984). In this approach, a 5'-O-DMT-protected mononucleotide is activated at the 3' position by, for example, chloro-(2-cyanoethoxy)-N,N-diisopropylaminophosphine. The resulting 5'-O-DMT-protected 3'-.beta.-cyanoethoxy-N,N-diisopropylamino phosphoramidite mononucleotide is reacted with the unprotected 5'-hydroxyl of an n-mer nascent oligonucleotide, resulting in condensation reaction and the formation of an (n+1)-oligomer.
Cao et at., Tetrahedron Lett. 24, 1019 (1983), offered an alternative to this approach. They reported activating the 5'-hydroxyl of the nascent oligonucleotide chain with methyl phosphoroditetrazolide (MPDT) and other heterocyclic bases. Reaction of 5'-DMT-protected mononucleosides having a free 3'-hydroxyl with the activated nascent oligonucleotide resulted in the addition of the mononucleoside to the oligonucleotide by formation of a methyl phosphite triester linkage. Oxidation with iodine in water led to oxidation of the methyl phosphite triester to a methyl phosphonate linkage.
The assembly of .sup.14 C- or .sup.3 H-labeled nucleoside phosphoramidite (3) has generally followed the method of Beaucage and Caruthers and requires a two-step process. (FIG. 1). E.g., Sasmor et al., J. Labeled Compd. and Radiopharm. 36, 15-31 (1995). Synthesis is accomplished by addition of the radiolabelled 3'-activated synthon (3) to a unprotected 5'-hydroxyl of a nascent oligonucleotide chain. Several disadvantages are associated with this method: (a) since the radioisotope is introduced in the very first step, the radiochemical yield after two steps is limited; (b) this operation often suffers a dilution problem, namely, the natural abundance isotope is usually blended in as a carrier in order to maintain a manageable synthetic scale, resulting in lower specific activity of the final oligos; (c) the phosphoramidite 3 is a reactive species prone to degradation--3 requires stringent storage and transportation conditions, and the degraded products from 3 could cause insufficient coupling when in use; (d) it is difficult to recover [.sup.3 H]- or [.sup.14 C]-3 intact after the coupling reaction. These drawbacks are costly, considering that in the current coupling protocol 3 and tetrazole are used at more than 10 times excess. Sasmor et al., supra.
Other methods of radiolabeling oligonucleotides, while avoiding the dilution problem, lead to indiscriminate labeling in multiple positions. E.g., Graham et al., Nucl. Acids Res. 21, 3737 (1993) and references cited therein. Still other methods employ radiolabeling at exchangeable positions, which magnifies the dilution problem. Graham et al., supra.
In view of the deficiencies in the prior art, improved methods of radiolabeling oligonucleotides are desirable.